BAG3 nucleotide and protein sequences to be used in research, diagnostics and therapy for cell death-involving diseases, and for modulation of cell survival and/or death

ABSTRACT

The present invention provides BAG3 nucleotide and protein sequences to be used in research, diagnostics and therapy for modulation of cell survival and/or death, in particular in leukemias, other neoplasias and apoptosis-involving diseases. More particularly the invention refers to the use of specific antisense-based constructs and peptide-specific polyclonal and monoclonal antibodies in leukemias, other neoplasias and cell death-involving diseases.

FIELD OF THE INVENTION

The present invention provides BAG3 nucleotide and protein sequences tobe used in research, diagnostics and therapy for cell death-involvingdiseases, and for modulation of cell survival and/or death.

More particularly the invention refers to the use of specificantisense-based constructs and peptide-specific polyclonal andmonoclonal antibodies in leukemias, other neoplasias and celldeath-involving diseases.

BACKGROUND

Cell death by apoptosis is largely responsible for control of tissuehomeostastis, differentiative and immune processes. Alterations in theapoptosis program are implied in acute and chronic tissue damages(heart, kidney, brain or other tissue ischaemia, chronic degenerativedisorders such as Parkinson's disease, amyotrophic lateral sclerosis andothers, etc.), characterized by excessive apoptosis, and neoplastic,autoimmune and other diseases involving insufficient apoptosis.Furthermore, since antineoplastic compounds mainly act by inducingapoptosis in cancer cells, molecules involved in the apoptotic responsedetermine neoplastic cell sensitivity or resistance to therapy.Biochemical components and/or regulators of the apoptotic pathways canbe targets for modulating therapies, some of which have shown efficacyin preclinical models and are now in human clinical trials. Furthermore,apoptosis-involved molecules can represent diagnostic tools in a rangeof diseases and reagents for laboratory work (1).

BAG3 is member of the BAG protein family, involved in co-chaperoneactivity for intracellular protein folding (2). Although BAG3 displayshomology with the other members of the BAG family in some portions, likethe BAG domain, other parts of its nucleotide and protein sequences areunique (2-4). These BAG3-specific, unique portions have been utilised byus for the invention here described.

In the following there are reported the BAG3 nucleotide and peptidesequences; the underlined parts correspond to parts which are consideredparticularly relevant for the present invention.

BAG3 nucleotide sequence (SEQ ID NO: 1):reference: NCBI PubMed, XM 055575Homo sapiens BCL2-associated athanogene 3 (BAG3), mRNAgi|16156810|ref|XM_(—)055575.1|[16156810]

   1 gcggagctcc gcatccaacc ccgggccgcg gccaactttt ttggactgga ccagaagttt  61 ctagccggcc agttgctacc tccctttatc tcctccttcc cctctggcag cgaggaggct 121 atttccagac acttccaccc ctctctggcc acgtcacccc cgcctttaat tcataaaggt 181 gcccggcgcc ggcttcccgg acacgtcggc ggcggagagg ggcccacggc ggcggcccgg 241 ccagagactc ggcgcccgga gccagcgccc cgcacccgcg ccccagcggg cagaccccaa 301 cccagcatga gcgccgccac ccactcgccc atgatgcagg tggcgtccgg caacggtgac 361 cgcgaccctt tgccccccgg atgggagatc aagatcgacc cgcagaccgg ctggcccttc 421 ttcgtggacc acaacagccg caccactacg tggaacgacc cgcgcgtgcc ctctgagggc 481 cccaaggaga ctccatcctc tgccaatggc ccttcccggg agggctctag gctgccgcct 541 gctagggaag gccaccctgt gtacccccag ctccgaccag gctacattcc cattcctgtg 601 ctccatgaag gcgctgagaa ccggcaggtg caccctttcc atgtctatcc ccagcctggg 661 atgcagcgat tccgaactga ggcggcagca gcggctcctc agaggtccca gtcacctctg 721 cggggcatgc cagaaaccac tcagccagat aaacagtgtg gacaggtggc agcggcggcg 781 gcagcccagc ccccagcctc ccacggacct gagcggtccc agtctccagc tgcctctgac 841 tgctcatcct catcctcctc ggccagcctg ccttcctccg gcaggagcag cctgggcagt 901 caccagctcc cgcgggggta catctccatt ccggtgatac acgagcagaa cgttacccgg 961 ccagcagccc agccctcctt ccaccaagcc cagaagacgc actacccagc gcagcagggg1021 gagtaccaga cccaccagcc tgtgtaccac aagatccagg gggatgactg ggagccccgg1081 cccctgcggg cggcatcccc gttcaggtca tctgtccagg gtgcatcgag ccgggagggc1141 tcaccagcca ggagcagcac gccactccac tccccctcgc ccatccgtgt gcacaccgtg1200 gtcgacaggc ctcagcagcc catgacccat cgagaaactg cacctgtttc ccagcctgaa1261 aacaaaccag aaagtaagcc aggcccagtt ggaccagaac tccctcctgg acacatccca1321 attcaagtga tccgcaaaga ggtggattct aaacctgttt cccagaagcc cccacctccc1381 tctgagaagg tagaggtgaa agttccccct gctccagttc cttgtcctcc tcccagccct1441 ggcccttctg ctgtcccctc ttcccccaag agtgtggcta cagaagagag ggcagccccc1501 agcactgccc ctgcagaagc tacacctcca aaaccaggag aagccgaggc tcccccaaaa1561 catccaggag tgctgaaagt ggaagccatc ctggagaagg tgcaggggct ggagcaggct1621 gtagacaact ttgaaggcaa gaagactgac aaaaagtacc tgatgatcga agagtatttg1681 accaaagagc tgctggccct ggattcagtg gaccccgagg gacgagccga tgtgcgtcag1741 gccaggagag acggtgtcag gaaggttcag accatcttgg aaaaacttga acagaaagcc1801 attgatgtcc caggtcaagt ccaggtctat gaactccagc ccagcaacct tgaagcagat1861 cagccactgc aggcaatcat ggagatgggt gccgtggcag cagacaaggg caagaaaaat1921 gctggaaatg cagaagatcc ccacacagaa acccagcagc cagaagccac agcagcagcg1981 acttcaaacc ccagcagcat gacagacacc cctggtaacc cagcagcacc gtagcctctg2041 ccctgtaaaa atcagactcg gaaccgatgt gtgctttagg gaattttaag ttgcatgcat2101 ttcagagact ttaagtcagt tggtttttat tagctgcttg gtatgcagta acttgggtgg2161 aggcaaaaca ctaataaaag ggctaaaaag gaaaatgatg cttttcttct atattcttac2221 tctgtacaaa taaagaagtt gcttgttgtt tcagaagttt aaccccgttg cttgttctgc2281 agccctgtct acttgggcac ccccaccacc tgttagctgt ggttgtgcac tgtcttttgt2341 agctctggac tggaggggta gatggggagt caattaccca tcacataaat atgaaacatt2401 tatcagaaat gttgccattt taatgagatg attttcttca tctcataatt aaaatacctg2461 actttagaga gagtaaaatg tgccaggagc cataggaata tctgtatgtt ggatgacttt2521 aatgctacat tttBAG3 aminoacidic sequence (SEQ ID NO: 2):reference: NCBI PubMed, XM 055575Homo sapiens BCL2-associated athanogene 3 (BAG3), mRNAgi|16156810|ref|XM_(—)055575.1|[16156810]

MSAATHSPMMQVASGNGDRDPLPPGWEIKIDPQTGWPFFVDHNSRTTTWNDPRVPSEGPKETPSSANGPSREGSRLPPAREGHPVYPQLRPGYIPIPVLHEGAENRQVHPFHVYPQPGMQRFRTEAAAAAPQRSQSPLRGMPETTQPDKQCGQVAAAAAAQPPASHGPERSQSPAASDCSSSSSSASLPSSGRSSLGSHQLPRGYISIPVIHEQNVTRPAAQPSFHQAQKTHYPAQQGEYQTHQPVYHKIQGDDWEPRPLRAASPFRSSVQGASSREGSPARSSTPLHSPSPIRVHTVVDRPQQPMTHRETAPVSQPENKPESKPGPVGPELPPGHIPIQVIRKEVDSKPVSQKPPPPSEKVEVKVPPAPVPCPPPSPGPSAVPSSPKSVATEERAAPSTAPAEATPPKPGEAEAPPKHPGVLKVEAILEKVQGLEQAVDNFEGKKTDKKYLMIEEYLTKELLALDSVDPEGRADVRQARRDGVRKVQTILEKLEQKAIDVPGQVQVYELQPSNLEADQPLQAIMEMGAVAADKGKKNAGNAEDPHTETQQPEATAAATSNPSSMTDTPGNPAAP

BAG3 protein is known to be expressed in some cell lines, such as HeLaand A2058, and, as far as normal primary human cells are concerned, inskeletal muscle, heart, ovary and other types of normal cells (2-5).BAG3 expression has also been detected in human pancreas tumour cells(6).

BAG3 expression had not been reported in other types of primary normalor neoplastic cells before the results here reported for the first time.

Some findings describe that transfection of cells of the human cell lineHeLa (5) or of the murine cell line 32D (7) with BAG3 hyperexpressingconstructs can modestly increase cell apoptosis induced by Baxmicroinjection or via Fas (5), or by IL-3 deprivation (7), respectively.

Generically antibodies for BAG3 have been described in WO00/14106 andWO95/25125, however there has not been characterized any immunogenicsite specific for them. Ref.s 4-6 describe polyclonal antibodiesspecific for the carbossi-terminal region of BAG3 protein starting fromamino acid 306 specifically. Liao describes a rabbit polyclonalanti-BAG3 antibody against the 196 amino acids of the C-terminal portionof BAG3. Lee describes a polyclonal antibody against the amino acidregion encompassing the portion 306-575. Dong describes a polyclonalantibody against the two amino acid regions 2 and 8.

Patent abstract of Japan publication 10327872 describes uses of BAG3 fordiagnosis, prophylaxis and therapy of pathologies relating to apoptosis,however there has not been characterized any immunogenic site or anyspecific antibody, moreover test, in particular in humans, are absent.

Before results here reported for the first time, BAG3 expression had notbeen proved to influence apoptosis in human primary cells, eithernormal, neoplastic or affected by other types of pathologies.Furthermore, BAG3 downmodulation by reagents, such as oligonucleotides,that can be used in primary cells, and its effects on cell apoptosis hadnever been reported.

SUMMARY OF THE INVENTION

The present invention refers to BAG3 protein (SEQ ID NO: 2) andcorresponding nucleotide sequence (SEQ ID NO: 1) and parts of them(indicated by underlining inside the above mentioned long sequences).

Objects of the present invention are therefore the uses of BAG3polypeptides and polynucleotides codifying it and parts of them inresearch, diagnostics and therapy for modulating primary cell survivaland/or death, particularly in human leukemias and other neoplasias orcell death-involving diseases.

There are considered within the scope of the invention in thatBAG3-related: sense or antisense oligonucleotides; monoclonal orpolyclonal antibodies that specifically recognise one or moreBAG3-specific epitopes: in particular:

SEQ ID NO 15: DRDPLPPGWEIKIDPQ; SEQ ID NO 16: SSPKSVATEERAAPS; SEQ ID NO17: DKGKKNAGNAEDPHT; SEQ ID NO 18: NPSSMTDTPGNPAAP;primers for PCR; nucleotide sequences for analysis of DNA or RNA; thepolypeptide and polynucleotide sequences encoding them, includingrecombinant DNA molecules, cloned genes or degenerate variants thereof,especially naturally occurring variants such as allelic variants.

Reagents and compositions for the uses described in the presentinvention additionally include vectors, including expression vectors,viruses, etc., containing BAG3-specific sequences; cells geneticallyengineered to contain such sequences and cells genetically engineered toexpress such sequences. Reagents additionally include the complement ofany of the nucleotide sequences recited above.

Compositions for the uses described in the present invention may furthercomprise an acceptable carrier, such as pharmaceutically acceptablecarrier.

BAG3-based uses described in the present invention include also methodsfor preventing, treating or ameliorating a medical condition, whichcomprises administering to a human or other animal subject atherapeutically effective amount of a composition comprising BAG3-basedreagents. Examples are methods for preventing, treating or ameliorating:acute or chronic tissue damages, such as heart, kidney, brain or otherorgan ischaemia, HIV-related damage of brain or other tissues, skeletalmuscle disorders, transplantation rejection; chronic degenerativedisorders such as Parkinson's disease, amyotrophic lateral sclerosis andothers, etc.; and neoplastic, autoimmune and other diseases involvingexcessive or defective apoptosis; tissue repair or wound healing,treatment of surgical incisions, and ulcers, such as stomach or diabeticulcers; etc.

BAG3-based uses described in the present invention relate also toreagents and methods for detecting the presence of BAG3 nucleotidesequence or protein, or parts of them. Such methods can, for example, beutilised as part of prognostic and diagnostic and/or prognosticevaluation of disorders as recited above and for the identification ofsubjects exhibiting a predisposition to such conditions. Furthermore,the invention include BAG3-related uses for evaluating the efficacy ofdrugs, and monitoring the progress of patients, involved in clinicaltrials for the treatment of disorders as recited above.

BAG3-related uses of the present invention include also reagents and/ormethods for the identification of compounds that modulate the expressionor the activity of BAG3. Such reagents or methods can be utilised, forexample, for the identification of compounds that can amelioratesymptoms of disorders as recited above. Such methods can include, butare not limited to, assays for identifying compounds and othersubstances that interact with (e.g., bind to) BAG3 protein or nucleotidesequence or parts of them.

The invention also includes methods for detecting the presence of thenucleotide sequence SEQ ID NO: 1 or of the protein SEQ ID NO: 2 or partsof them in a sample, in particular at least a part identified as SEQ IDNO: 3, 4, 5, 6, 7, 8, 15, 16, 17, 18; said method comprising the stepsof: contacting the sample with a compound that binds to and forms acomplex with the nucleotide or the protein in sufficient conditions toform the complex, and detecting said complex. The expert in the field isable to select the suitable conditions to perform the method.

The invention also includes methods for detecting a compound that bindsto the protein SEQ ID NO: 2 or parts of it in a sample, in particular atleast a part identified as SEQ ID NO: 4, 6, 8, 15, 16, 17, 18; saidmethod comprising the steps of: contacting the compound with the proteinor its part/s in sufficient conditions to form the complexcompound/protein or its part/s, and detecting said complex. The expertin the field is able to select the suitable conditions to perform themethod. The invention also includes methods for the treatment ofdisorders as recited above which may involve the administration of suchcompounds to individuals exhibiting symptoms or tendencies related todisorders as recited above. In addition, the invention encompassesmethods for treating diseases or disorders as recited above byadministering compounds and other substances that modulate the overallactivity of BAG3 and related molecules. Compounds and other substancescan effect such modulation either on the level of gene expression orprotein activity.

The diagnostic, prognostic or therapeutic compositions for theBAG3-related use related to the present invention are also presentlyvaluable for veterinary applications. Particularly domestic animals andthoroughbred horses, in addition to humans, are desired patients forsuch applications.

The invention further refers to a kit for identification and diagnosiscomprising the polyclonal or monoclonal antibodies identified in thefollowing description or nucleotide sequence SEQ ID NO: 1 or the proteinSEQ ID NO: 2 or parts of them, in particular at least a part identifiedas SEQ ID NO: 3, 4, 5, 6, 7, 8, 15, 16, 17, 18; or the antisense andnonsense oligos identified as SEQ ID NO: 9, 10, 11, 12, 13, 14, orfunctionally equivalents of the above identified sequences.

BAG3-based uses described in the present invention relate also toreagents and/or methods and/or kits for laboratory work or research.

Further objects of the invention will become evident from the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression of BAG3 mRNA (A panel) and protein (B panel)in primary cells from leukemia patients.

FIG. 2 shows the BAG3 downmodulation ability of anti-BAG3 antisenseoligodeoxynucleotides in primary cells from leukemia patients.

FIG. 3 shows the stimulation of mitochondrial cytochrome c release byanti-BAG3 antisense oligodeoxynucleotides in primary cells from leukemiapatients.

FIG. 4 shows the stimulation of caspase activity by anti-BAG3 antisenseoligodeoxynucleotides in primary cells from leukemia patients.

FIG. 5 shows the enhancement on annexin V binding by anti-BAG3 antisenseoligodeoxynucleotides in primary cells from leukemia patients.

FIG. 6 shows the stimulation of primary B-CLL (B chronic lymphocyticleukemia) cell apoptosis by anti-BAG3 antisense oligodeoxynucleotides.

FIG. 7 shows the stimulation of primary ALL (acute lymphoblasticleukemia) cell apoptosis by anti-BAG3 antisense oligodeoxynucleotides.

FIG. 8 shows the BAG3 downmodulation ability of anti-BAG3 antisenseoligodeoxynucleotides in human U937 cells.

FIG. 9 shows the stimulation of stress-induced apoptosis in cells of thehuman myeloid leukemia line U937 by anti-BAG3 antisenseoligodeoxynucleotides.

FIG. 10 shows the stimulation of stress-induced apoptosis in humannormal peripheral blood primary lymphocytes (A panel) or monocytes (Bpanel) by anti-BAG3 antisense oligodeoxynucleotides.

FIG. 11 shows the expression of BAG-3 protein and its modulation byantisense oligonucleotides, as detected in Western blotting (A) orintracellular immunofluorescence (B).

FIG. 12 shows the effect of BAG3-specific antisense oligonucleotides orAraC on ALL cell apoptosis.

Table 1 describes the effect of anti-BAG3 antisenseoligodeoxynucleotides on apoptosis in cells of the human osteosarcomaline SAOS.

Table 2 describes the protective effect of BAG3 hyperexpression onstress-induced apoptosis in the human cell line 293.

Table 3 describes the effect of BAG3 hyperexpression on the growth ofhuman neoplastic (osteosarcoma) cells xenografted in nude mice.

Table 4 shows the results of the ELISA tests performed to verify thebinding of hybridoma mother clone supernatants to MAP-BAG3 constructs.

FIG. 13 shows the binding of the polyclonal antibodies AC-BAG3-2 andAC-BAG3-1 to lysates from HeLa or primary leukemia cells (A) and of thehybridoma mother clones (AC-1, AC-2, AC-3, AC-4) supernatants to celllysates from HeLa cells (B), as detected by Western blotting.

DETAILED DESCRIPTION OF THE INVENTION

The polynucleotidic and aminoacidic fragments that are consideredparticularly relevant for the present invention and are comprised insideSEQ ID NO: 1 and 2, are indicated in the following, such sequences arerelevant because are specific of BAG3 and not shared with any otherknown sequence of other BAG genes or proteins:

SEQ ID NO: 3: gcggagctcc gcatccaacc ccgggccgcg gccaactttt ttggactggaccagaagttt ctagccggcc agttgctacc tccctttatc tcctccttcc cctctggcagcgaggaggct atttccagac acttccaccc ctctctggcc acgtcacccc cgcctttaattcataaaggt gcccggcgcc ggcttcccgg acacgtcggc ggcggagagg ggcccacggcggcggcccgg ccagagactc ggcgcccgga gccagcgccc cgcacccgcg ccccagcgggcagaccccaa cccagcatga gcgccgccac ccactcgccc atgatgcagg tggcgtccggcaacggtgac SEQ ID NO: 4: MSAATHSPMMQVASGNGDRDPLPPGWEIKIDPQTG SEQ ID NO:5: gtgcc ctctgagggc cccaaggaga ctccatcctc tgccaatggc ccttcccgggagggctctag gctgccgcct gctagggaag gccaccctgt gtacccccag ctccgaccaggctacattcc cattcctgtg ctccatgaag gcgctgagaa ccggcaggtg caccctttccatgtctatcc ccagcctggg atgcagcgat tccgaactga ggcggcagca gcggctcctcagaggtccca gtcacctctg cggggcatgc cagaaaccac tcagccagat aaacagtgtggacaggtggc agcggcggcg gcagcccagc ccccagcctc ccacggacct gagcggtcccagtctccagc tgcctctgac tgctcatcct catcctcctc ggccagcctg ccttcctccggcaggagcag cctgggcagt caccagctcc cgcgggggta catctccatt ccggtgatacacgagcagaa cgttacccgg ccagcagccc agccctcctt ccaccaagcc cagaagacgcactacccagc gcagcagggg gagtaccaga cccaccagcc tgtgtaccac aagatccagggggatgactg ggagccccgg cccctgcggg cggcatcccc gttcaggtca tctgtccagggtgcatcgag ccgggagggc tcaccagcca ggagcagcac gccactccac tccccctcgcccatccgtgt gcacaccgtg gtcgacaggc ctcagcagcc catgacccat cgagaaactgcacctgtttc ccagcctgaa aacaaaccag aaagtaagcc aggcccagtt ggaccagaactccctcctgg acacatccca attcaagtga tccgcaaaga ggtggattct aaacctgtttcccagaagcc cccacctccc tctgagaagg tagaggtgaa agttccccct gctccagttccttgtcctcc tcccagccct ggcccttctg ctgtcccctc ttcccccaag agtgtggctacagaagagag ggcagccccc agcactgccc ctgcagaagc tacacctcca aaaccaggagaagccgaggc tcccccaaaa catccaggag SEQ ID NO: 6:NDPRVPSEGPKETPSSANGPSREGSRLPPAREGHPVYPQLRPGYIPIPVLHEGAENRQVHPFHVYPQPGMQRFRTEAAAAAPQRSQSPLRGMPETTQPDKQCGQVAAAAAAQPPASHGPERSQSPAASDCSSSSSSASLPSSGRSSLGSHQLPRGYISIPVIHEQNVTRPAAQPSFHQAQKTHYPAQQGEYQTHQPVYHKIQGDDWEPRPLRAASPFRSSVQGASSREGSPARSSTPLHSPSPIRVHTVVDRPQQPMTHRETAPVSQPENKPESKPGPVGPELPPGHIPIQVIRKEVDSKPVSQKPPPPSEKVEVKVPPAPVPCPPPSPGPSAVPSSPKSVATEERAAPSTAPAEATPPKPGEAEAPPKHPGVLKVEAILEKVQGLEQAVDNFEG SEQ ID NO: 7 attgatgtcccaggtcaagt ccaggtctat gaactccagc ccagcaacct tgaagcagat cagccactgcaggcaatcat ggagatgggt gccgtggcag cagacaaggg caagaaaaat gctggaaatgcagaagatcc ccacacagaa acccagcagc cagaagccac agcagcagcg acttcaaaccccagcagcat gacagacacc cctggtaacc cagcagcacc gtagcctctg ccctgtaaaaatcagactcg gaaccgatgt gtgctttagg gaattttaag ttgcatgcat ttcagagactttaagtcagt tggtttttat tagctgcttg gtatgcagta acttgggtgg aggcaaaacactaataaaag ggctaaaaag gaaaatgatg cttttcttct ataftcttac tctgtacaaataaagaagtt gcttgttgtt tcagaagttt aaccccgttg cttgttctgc agccctgtctacttgggcac ccccaccacc tgttagctgt ggttgtgcac tgtcttttgt agctctggactggaggggta gatggggagt caattaccca tcacataaat atgaaacatt tatcagaaatgttgccattt taatgagatg attttcttca tctcataatt aaaatacctg actttagagagagtaaaatg tgccaggagc cataggaata tctgtatgtt ggatgacttt aatgctacat tttSEQ ID NO: 8: ELQPSNLEADQPLQAIMEMGAVAADKGKKNAGNAEDPHTETQQPEATAAATSNPSSMTDTPGNPAAP

The experiments performed in our laboratories indicate for the firsttime that specific antisense oligonucleotides are able to modulate, inhuman primary cells and human cell lines, the levels of BAG3 protein;these antisense oligos modulate also the survival and/or death, eitherspontaneous or in response to therapy, of human primary cells and humancell lines. Experiments with primary cells, that are the target ofdiagnostic and therapeutic applications, are particularly relevant, andthe results were not predictable from data obtained with cell lines,since stable cell lines and primary cells are differently sensitive tomodulators of cell survival and/or death (14-18); furthermore, theeffect of BAG3 protein downmodulation on cell survival and/or death,either in cell lines or primary cells, were not reported before, norwere predictable from data concerning BAG3 hyperexpression, sinceseveral examples have been reported, in which the overexpression of aprotein (i.e. Bcl-2 family proteins) can protect cells frompro-apoptotic insults, but its downmodulation does not stimulateapoptosis (19-21); finally, BAG3 downmodulation has been obtained withspecific antisense oligonucleotides, that can be used for research,diagnosis and/or therapy, and their effectiveness was not predictablebefore the experimental work, since not all antisense oligonucleotidesagainst a specific mRNA display comparable activities when introduced ina cell, and furthermore some antisense molecules can exert unpredicted,not desired effects, such as citotoxicity (22-23).

BAG3 modulation is able to influence the development of a human tumourin vivo; these results are necessary for in vivo applications, areabsolutely required for proving the biological activity of a gene and/orprotein and the effects of its modulation in pluricellular organisms,and cannot be extrapolated in this respect from results in vitro (1).

Based on the apoptosis-modulating effect of the antisense according tothe invention, a panel of polyclonal and monoclonal antibodies raisedagainst peptide constructs (MAP-BAG3-peptides) has been designed to: mapdifferent BAG3 epitopes and/or domains; relate them to the functionalactivity of BAG3 (i.e., modulation of cell survival); relate them tospecific biochemical interaction with molecular partners and/orformation of complexes; target them to neutralize (antagonisticantibodies) or trigger (agonistic antibodies) BAG3 functional activity.

Identification of BAG3 Expression in Human Primary Leukemia Cells andEffectiveness of Specific Antisense Oligonucleotides in Modulating BAG3Levels and Cell Survival and/or Death.

We analysed by PCR the expression of BAG3 mRNA in primary cells fromB-CLL patients. BAG3 mRNA was detectable in such cells, and its levelsappeared to be enhanced by treatment with a chemotherapeutic compound,fludarabine phosphate (FIG. 1, A panel).

-   -   To explore the levels of BAG3 protein, we first used a        polyclonal antibody according to the teaching of the patent appl        WO95/25125. Such antibody appeared to bind with a low avidity        BAG3 protein from primary leukemic cells and had therefore to be        used in condition of high resolution (high antibody        concentration, long incubation times, etc.). Therefore we        decided to produce novel polyclonal antibodies by using a        different approach, i.e. using a Multiple Antigen Peptide (MAP)        prepared in a single synthesis by the solid-phase method        described in ref. 24-26. Such approach allows to improve the        immunogenecity of the antigenic peptides and obtain particularly        efficient antibodies. (24-26) This is of high relevance for        detecting proteins expressed in low amounts, as usually happens        for many relevant proteins in physiologic or pathologic        conditions in primary cells. The kind of MAP used, here as for        the subsequent production of hybridomas (see below), was an        octa-branching MAP consisting of a core matrix made up of three        levels of lysine and eight amino terminals for anchoring peptide        antigens. In this case, we used the peptide NPSSMTDTPGNPAAP (SEQ        ID NO: 18), corresponding to the last 15 aminoacids of the        carboxyterminal region of BAG3 protein. For obtaining policlonal        antibodies, two rabbits were immunised with 4 boosts (a boost        every 2 week) of MAP-BAG3-4 (400 micrograms for each boost); the        serum was finally tested against MAP-BAG3-4 in ELISA test and        verified to be positive. We named the two polyconal antibodies,        obtained from the two rabbits, AC-BAG3-1 and AC-BAG3-2: both        recognised the carboxyterminal region of BAG3 protein and were        efficient in detecting BAG3 protein, either in Western blotting        or in immunofluorescence, in primary cells, as shown in FIGS.        1,2,11,13.    -   With this BAG3-specific antibodies we analysed by        immunofluorescence the expression of BAG3 protein, that was        detectable in primary cells from B-CLL patients and whose levels        appeared to be enhanced by treatment fludarabine (FIG. 1, B        panel). In a comprehensive investigation of 18 different B-CLL        specimens, 13 displayed detectable levels of BAG3 protein, and        in 11 of these BAG3 levels were upregulated by treatment with        fludarabine.

These findings for the first time demonstrate that BAG3 expression canbe detected in primary leukemic cells and modulated by therapy. Suchresults disclose a diagnostic and/or prognostic use, not shown before,of BAG3-detecting reagents in leukemias.

To be able to modulate BAG3 expression, we constructed the followingBAG-3-based antisense oligonucleotides:

antisense 1: TGCATCATGG GCGAGTGGGT GGCGG, (SEQ ID NO: 9) antisense 2:GCTCATGCTG GGTTGGGGTC TG, (SEQ ID NO: 10) antisense 3: ATTAAAGGCGGGGGTGACGT GG, (SEQ ID NO: 11)and control nonsense:

nonsense 1: TTATATTCTATTATATTTATGAACTCC, (SEQ ID NO: 12) nonsense 2:CCTCGTAACCACCG ACCTCAAT, (SEQ ID NO: 13) nonsense 3: GCTTATGGAGGATTGAGGTT GG. (SEQ ID NO: 14)

Other oligonucleotides can be constructed, functionally analogues to theones mentioned before, in particular the oligonucleotides can beconstructed based on sequences indicated as SEQ ID NO: 3, 5, 7.

There are within the scope of the present invention the nucleotide andpeptide sequences that show functional equivalence with the onesidentified in the description or that have a homology of at least 75%,preferably at least 80% homology, more preferably at least 90% homology,more preferably at least 95% homology, even more preferably at least 98%homology to at least one of the sequences mentioned in the description.

Administering of antisense, but not of nonsense, oligonucleotides tohuman primary leukemic cells ex vivo resulted in a downmodulation ofBAG3 protein levels. Representative results are shown in FIG. 2;analogous results were obtained in experiments with three differentB-CLL specimens. These findings disclose the use, not shown before, ofBAG3 antisense oligonucleotides for affecting BAG3 protein levels inprimary (in this case neoplastic, and specifically leukemic) cells. Wethen analysed whether antisense oligonucleotides, by downmodulating BAG3protein levels, could affect cell apoptosis. Primary cells from leukemiapatients were incubated with or without antisense or controloligonucleotides and/or fludarabine, and different events of apoptosis:mitochondrial cytochrome c release (8), caspase 3 activation (9),annexin V binding (10) and appearance of hypodiploid elements (11) wereanalysed. A comprehensive analysis of 15 B-CLL samples indicated thatadministering of antisense, but not of nonsense, oligonucleotides to thecells resulted in stimulation of mitochondrial cytochrome c release(FIG. 3), caspase activity (FIG. 4), annexin V binding (FIG. 5) andappearance of hypodiploid elements (FIG. 6). Apoptosis stimulation waseven more amplified by the addition of fludarabine (FIG. 6).Furthermore, in 4 of 4 ALL specimens analysed, the pro-apoptotic effectof the antisense oligonucleotides alone was particularly remarkable,since the percentage of hypodiploid elements reached >60% of the cells(similar to the value obtained with the chemotherapeutic compound AraC)(FIG. 7).

Therefore we demonstrate for the first time that downmodulation of BAG3protein levels by administration of BAG3 antisense oligonucleotides todifferent types of human primary leukemia cells can stimulate apoptosis.The pro-apoptotic effect is remarkable when the antisenseoligonucleotides are administered alone and can be synergic with that ofdifferent chemotherapeutic compounds.

These findings disclose the possible use, not shown before, ofBAG3-modulating reagents, such as antisense oligonucleotides, formodulating survival and/or death in human primary cells, in this caseneoplastic, and specifically leukemic. They also indicate the possibleuse of such reagents in synergy with other drugs. Additional resultswere obtained by using human cells of different types: osteosarcomacells of the SAOS line, in which we detected a remarkable pro-apoptoticeffect of the antisense oligonucleotides alone (table 1); and myeloidcells of the U937 line, in which BAG3 antisense could enhance apoptosisinduced by stress (FIG. 9). Particularly, the enhancement ofstress-induced apoptosis in U937 cells suggested to us to verify whetherBAG3-based reagents could interfere also with stress effects in humanprimary cells. Therefore we administered the antisense or controloligonucleotides to human normal peripheral blood lymphocytes ormonocytes ex vivo, treated with the stress inducers diethylmaleate (DEM)and 2-Methoxymethylestradiol (2-ME). Antisense, but not control, oligo,highly enhanced cell apoptosis in these cells (FIG. 10). These findingsfor the first time demonstrate that stress effects on human primarycells (in this case, normal cells, and specifically lymphocytes andmonocytes from peripheral blood) can be modulated by BAG3-basedreagents.

We investigated whether protection from cell death could be obtainedwith BAG3-based reagents. Therefore we transfected 293 cells with aBAG3-hyperexpressing construct and verified the effect on stress-inducedapoptosis. Transfection with the BAG3 construct resulted in protectionfrom stress-induced apoptosis (table 2).

The above described results indicate for the first time that: 1) BAG3 isexpressed in human primary leukemic cells; 2) BAG3 protein levels, andspontaneous or therapy-induced death of human primary cells, can bemodulated by using specific antisense oligonucleotides.

It is worthy of note that this is the first reported observation thatspecific BAG3 antisense oligonucleotides are able to enhance humanprimary cell apoptosis. It has been previously shown that theoverexpression of BAG-3 in transfected cell lines could partiallyprotect them from apoptosis induced via Fas or growth factor deprivation(5,7). Our invention was not predictable from such previous observation,for three reasons: 1) stable cell lines and primary cells aredifferently sensitive to modulators of cell survival and/or death(14-18); 2) several examples have been reported, in which theoverexpression of a protein (i.e. Bcl-2 family proteins) can protectcells from pro-apoptotic insults, but its downmodulation does notstimulate apoptosis (19-21); 3) not all antisense oligonucleotidesagainst a specific mRNA display comparable activities when introduced ina cell; furthermore, some antisense molecules can exert unpredicted, notdesired effects, such as citotoxicity (22-23). Therefore, the propertiesof the specific antisense oligonucleotide sequences used by us could notbe predicted before our experimental work.

This is the first reported observation of BAG3 expression in humanprimary leukemic cells. This was not predictable from previous resultsdescribed in stable cell lines and primary cells other than leukemiacells. Indeed: a) cell lines are no longer subjected to theenvironmental influences of a pluricellular organisms, and furthermoreand more importantly are selected for their survival in culture:therefore they usually differ in gene expression and/or levels ofparticular proteins from primary cells, even when belonging to the sametype; b) different cell types, either from lines or primary cells,differ in gene expression and/or levels of particular proteins (14-19).

Finally, since we have demonstrated that the modulation of BAG3 proteinlevels can modulate cell survival and/or death in primary cells, alsopolynucleotides and corresponding codified polypeptides indicated as SEQID NO: 2, 3, 4, 5, 6, 7, 8, 15, 16, 17, 18 and constructs comprisingthem dare una definizione minima di costrutto (hyperexpressingconstructs, either in plasmid or other vectors; naked DNA; etc.) thatpositively modulate such levels are relevant in this functionalactivity. Particularly, we have demonstrated that the functional effectis specific of BAG3 and not shared with other BAG proteins, andtherefore the SEQ ID NO 3, 5 and 7 are identified as particularlyrelevant for the functional effect (i.e., modulation of cell survivaland/or death).

Demonstration that BAG3 Modulation can Influence Tumour Development InVivo.

Hyperexpression of BAG3 had been reported to suppress apoptosis in celllines in vitro, since the overexpression of BAG-3 in transfected celllines could partially protect them from apoptosis induced via Fas orgrowth-factor deprivation (5,7). These results did not allow to predictthe effect of BAG3 hyperexpression on tumour development in vivo.Indeed, tumours in vivo are subjected to the environmental influences ofa pluricellular organisms, and molecules that have a specific activityin vitro can fail their effects or show different activities in vivo:therefore effects in vivo cannot be extrapolated from results in vitroand a specific experimental work in vivo is required (1). We transfectedcells of the human osteosarcoma cell line Saos with aBAG3-overexpressing plasmid vector and obtained a mass culture of stablytransfected cells. Wild type cells, the transfected culture and acontrol, void vector-transfected culture were injected in threedifferent sites (back, left and right sides) in nude mice. Wild type andcontrol cells did not give rise to any tumour, whileBAG3-hyperexpressing cells developed detectable tumours, demonstratingthat BAG3 modulation can influence the development of a tumour in vivo(table 3).

Design and Construction of a Panel of Polyclonal and MonoclonalAntibodies

The antibodies were raised against peptide constructs(MAP-BAG3-peptides) to recognise and/or trigger the following definedBAG3 epitopes and/or domains

SEQ ID NO 15: DRDPLPPGWEIKIDPQ; SEQ ID NO 16: SSPKSVATEERAAPS; SEQ ID NO17: DKGKKNAGNAEDPHT; SEQ ID NO 18: NPSSMTDTPGNPAAPof functional importance in human primary cells and other cell types ofdifferent origins.

The above described antisense oligonucleotides were all able todownmodulate the levels of BAG3 protein. This is relevant for theconsequent modulation of cell death, here reported for the first time.

The functional activity of BAG3 in modulating cell survival and/or deathcan rely on biochemical interactions of specific BAG3 epitopes and/ordomains indicated as SEQ ID NO 16, 16, 17, 18 with molecular partnersinvolved in survival/death pathway (2). Indeed, a variety of suchpartners have been detected for BAG proteins in general and BAG3 inparticular (2-5); such interactions can potentially involve differentparts of the molecule, such as, in addition to the BAG domain, the WWdomain, the SER-rich part, the PRO-rich part, etc. (described in 2-5).In view of the functional activity of BAG3 in modulating cell survival,it is important to be able to map different BAG3 epitopes and/ordomains: this can allow to: 1) relate such epitopes to the functionalactivity of BAG3; 2) identify the site(s) of interactions with knownpartners, as well as new sites of interactions with still undescribedpartners; 3) interfere with the formation of complexes with molecularpartners; 4) block or mimic the interaction with these partners, leadingto modulation of BAG3 functional activity.

To produce effective tools able to explore the above mentioned issues,we decided to obtain polyclonal and monoclonal antibodies againstspecific peptides representing spatially distinct portions of BAG3protein. Such polyclonal and monoclonal antibodies are desirable to: mapdifferent BAG3 epitopes and/or domains; relate them to the functionalactivity of BAG3 (i.e., modulation of cell survival); relate them tospecific biochemical interaction with molecular partners and/orformation of complexes; target them to neutralize (antagonisticantibodies) or trigger (agonistic antibodies) BAG3 functional activity.

We identified the following, spatially distinct BAG3-derived peptides:

(SEQ ID NO 15): DRDPLPPGWEIKIDPQ; (SEQ ID NO 16): SSPKSVATEERAAPS; (SEQID NO 17): DKGKKNAGNAEDPHT;

(SEQ ID NO 18): NPSSMTDTPGNPMP. Such peptide corresponded to one we usedfor raising the polyclonal antibodies (see above). Its use is here aimedat obtaining monoclonal antibodies against the carboxyterminal part ofBAG3 (indeed, only polyclonal antibodies against such part have been sofar: see ref. 4-6). Furthermore, its use is in addition, and notalternative, to that of the other three peptides (SEQ ID N. 15, 16 and17).

We used these peptides to obtain separate monoclonal antibodies againsteach one of the four peptides.

All four peptides are specific of BAG3 protein and are not shared withother any protein, including other BAG proteins.

For immunizing the animals, we decided to use MAPs (Multiple AntigenicPeptides) (24-26). As described in the previous section, theconstruction of MAPs allows to significantly enhance the immunogenicityof the antigenic peptides and to obtain particularly efficientantibodies. This is of relevance for detecting proteins expressed in lowamounts, as usually happens for many relevant proteins in physiologic orpathologic conditions in primary cells. Following this approach, weobtained the following unique map constructs:

(SEQ ID NO 15) MAP-BAG3-1: nh2-DRDPLPPGWEIKIDPQ-MAP containing (SEQ IDNO 16) MAP-BAG3-2: nh2-SSPKSVATEERAAPS-MAP containing (SEQ ID NO 17)MAP-BAG3-3: nh2-DKGKKNAGNAEDPHT-MAP containing (SEQ ID NO 18)MAP-BAG3-4: nh2-NPSSMTDTPGNPAAP-MAP containing

The production of the polyclonal antibodies has been described above. Inthis respect, as well as for the production of monoclonal antibodies,MAP constructs are to be considered unique and different from the simplepeptides alone, since their ability to elicit immunogenic responses inthe animal is different form that of the peptides used alone (24-26).

Monoclonal antibodies (not yet reported in literature) were highlyrequired, either in general because of the high specificity andhomogeneity of such reagents, but also in particular in view of ourresults demonstrating the apoptosis-modulating properties of BAG3protein in primary cells. Indeed BAG proteins, including BAG3, interactwith several molecular partners (2-5,7), and monoclonal antibodies arerequired to map the protein epitopes involved in interacting withspecific partners, thereby leading to effects on cell survival and/ordeath. Furthermore, monoclonal antibodies can display agonistic orantagonistic properties respect to the biological functions of aprotein, and this is of relevance for the potential application inmodulating BAG3 activity in cell survival and/or death.

For obtaining the monoclonal antibodies, we followed standardprocedures, already performed in our laboratory (12). Specifically:

-   -   nine Balb/c female mice of 4 weeks were immunised with 4 boosts        (a boost every 2 week) of the four MAP-BAG3 together (200        micrograms each, i.e. 800 micrograms of total        protein/mouse/boost). Spleens were then obtained and fused with        myeloma cells (NS0) to obtain monoclonal antibodies mother        clones. These were tested against each of the four MAP-BAG3 in        ELISA test (see table 4).

We produced:

-   -   nine murine monoclonal antibody mother clones (AC-1, AC-2, AC-3,        AC-4, AC-5, AC-6, AC-7, AC-8, AC-9) obtained from mice immunised        with the four MAP-BAG3 together. The nine mother clones are        presently being subcloned to obtain hybridomas against each one        of the four MAP-BAG3 constructs. The ELISA tests of the        antibodies produced by the nine mother clones are presented in        table 4. Importantly, the ELISA tests demonstrate that the        mother clones contains hybridomas able to recognise each one of        the four MAP-BAG3 used. Therefore, the nine mother clones        already contain several specific hybridomas, each of whom can        recognise one of the four epitopes represented in the MAPs and        can hence be used to map one BAG3 epitope and interfere with its        functional interactions and activities; the monospecific        hybridomas are presently being separated by subcloning        procedures.    -   The detection, by Western blot analysis, of BAG3 protein in        lysates from the cell line HeLa and primary leukemia cells are        shown in FIG. 13. Specifically:    -   the antibodies from the nine monoclonal mother clones did        recognise the four MAP-BAG3 constructs in ELISA test (table 4);    -   four of them have been as yet tested, with positive results, in        Western blot with HeLa lysates (FIG. 13, B panel).

In conclusion, the nine murine monoclonal antibody mother clones (AC-1,AC-2, AC-3, AC-4, AC-5, AC-6, AC-7, AC-8, AC-9) contain hybridomasspecific for each one of the four MAP-BAG3 constructs, and are able toidentify spatially distinct parts of BAG3 molecule, in particular themother clone AC-1 was no PD02009 deposited on the 17/12/2002 at theCentro Biotecnologie Avanzate di Genova They can therefore be used to:map different BAG3 epitopes and/or domains; relate them to thefunctional activity of BAG3 (i.e., modulation of cell survival); relatethem to specific biochemical interaction with molecular partners and/orformation of complexes; target them to neutralize (antagonisticantibodies) or trigger (agonistic antibodies) BAG3 functional activity.

The original features of these results are:

-   -   the downmodulating effect of antisense oligos constitutes the        original rationale, not predictable before, leading to the        necessity of mapping and triggering BAG3 epitopes that mediate        the mechanism of apoptosis modulation. This constituted the        premise for the production of a panel of antibodies        raised-against different region of BAG3 protein;    -   a panel of nine monoclonal-producing mother clones (AC-1; AC-2;        AC-3; AC-4; AC-5; AC-6; AC-7; AC-8; AC-9) have been obtained and        can be used to: map different BAG3 epitopes and/or domains;        relate them to the functional activity of BAG3 (i.e., modulation        of cell survival); relate them to specific biochemical        interaction with molecular partners and/or formation of        complexes; target them to neutralize (antagonistic antibodies)        or trigger (agonistic antibodies) BAG3 functional activity;    -   two polyclonal antibodies (AC-BAG3-1 and AC-BAG3-2), able to        reveal the presence of BAG3 protein in human primary leukemias        and its modulated expression of BAG3 protein by specific        antisense oligodeoxynucleotides, have been obtained.

Within the scope of the present invention, BAG3-protein, thecorresponding polynucleotide, corresponding parts of them andcorresponding antisense oligonucleotides can be used for research,diagnostic and therapeutic purposes for example in leukemias, otherneoplasias and cell death-involving diseases, and for modulation of cellsurvival and/or death. BAG3-based reagents include in a non-limitativemanner, oligonucleotides, primers, probes, (poly)peptides or protein,polyclonal or monoclonal antibodies, etc., and any other reagent able todetect or modulate BAG3 expression.

Findings illustrated in the present invention and obtained with thedescribed BAG3-based reagents could be obtained with modified reagentswith equivalent activities. These latter are therefore consideredequivalent to those illustrated in the present invention.

Particularly, as far as protein or its parts, or peptides, areconcerned, are considered equivalent:

-   -   naturally occurring (poly)peptides or proteins, that are        (poly)peptides or proteins produced by cells that have not been        genetically engineered and specifically contemplates various        (poly)peptides or proteins arising from post-translational        modifications of the (poly)peptide or protein including, but not        limited to, acetylation, carboxylation, glycosylation,        phosphorylation, lipidation and acylation;    -   derivatives, that are (poly)peptides or proteins chemically        modified by such techniques as ubiquitination, labeling (e.g.,        with radionuclides, fluorochromes or various enzymes),        pegylation (derivatization with polyethylene glycol) and        insertion or substitution by chemical synthesis of amino acids        such as ornithine, which do not normally occur in human        proteins;    -   recombinant variants, that are (poly)peptides or proteins        differing from naturally occurring (poly)peptides or proteins by        amino acid insertions, deletions, and substitutions, created        using recombinant DNA techniques. Guidance in determining which        amino acid residues may be replaced, added or deleted without        abolishing activities of interest, such as cellular trafficking,        may be found by comparing the sequence of the particular        polypeptide with that of homologous peptides and minimizinig the        number of amino acid sequence changes made in regions of high        homology.

Preferably, amino acid substitutions are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. The variationallowed may be experimentally determined by systematically makinginsertions, deletions, or substitutions of amino acids in a polypeptidemolecule using recombinant DNA techniques and assaying the resultingrecombinant variants for activity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered (poly)peptides or proteins. Such alterations can, for example,alter one or more of the biological functions or biochemicalcharacteristics. For example, such alterations may change (poly)peptideor protein characteristics such as ligand-binding affinities, interchainaffinities, or degradation/turnover rate. Further, such alterations canbe selected so as to generate (poly)peptides or proteins that are bettersuited for expression, scale up and the like in the host cells chosenfor expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

Substantially equivalent can be either nucleotide or amino acidsequences, for example a mutant sequence, that varies from a referencesequence by one or more substitutions, deletions, or additions, the neteffect of which does not result in an adverse functional dissimilaritybetween the reference and subject sequences. Typically, such asubstantially equivalent sequence varies from the reference one by nomore than about 20%, i.e. the number of individual residuesubstitutions, additions, and/or deletions in a substantially equivalentsequence; as compared to the corresponding reference sequence, dividedby the total number of residues in the substantially equivalent sequenceis about 0.2 or less. Such a sequence is said to have 80% sequenceidentity to the listed sequence. In one embodiment, a substantiallyequivalent, mutant, sequence of the invention varies from a listedsequence by no more than 10% (90% sequence identity); in a variation ofthis embodiment, by no more than 5% (95% sequence identity); and in afurther variation of this embodiment, by no more than 2% (98% sequenceidentity). Compared to aminoacidic identity, substantially equivalentnucleotide sequence(s) of the invention can have lower percent sequenceidentities, taking into account, for example, the redundancy ordegeneracy of the genetic code. For the purposes of the presentinvention, sequences having substantially equivalent biological activityand substantially equivalent expression characteristics are consideredsubstantially equivalent. For the purposes of determining equivalence,truncation of the mature sequence (e.g., via a mutation, which creates aspurious stop codon) should be disregarded.

Nucleic acid sequences encoding such substantially equivalent sequences,sequences of the recited percent identities can routinely be isolatedand identified via standard hybridization procedures well known to thoseof skill in the art.

Where desired, an expression vector may be designed to contain a signalor leader sequence which will direct the polypeptide through themembrane of a cell. Such a sequence may be naturally present or providedfrom heterologous protein sources by recombinant DNA techniques.

Recombinant variants encoding these same or similar (poly)peptides orproteins may be synthesized or selected by making use of the redundancyin the genetic code. Various codon substitutions, such as the silentchanges, which produce various restriction sites, may be introduced tooptimize cloning into a plasmid or viral vector or expression in aparticular prokaryotic or eukaryotic system. Mutations in thepolynucleotide sequence may be reflected in the polypeptide or domainsof other peptides added to the polypeptide to modify the properties ofany part of the polypeptide, to change characteristics such asligand-binding affinities, interchain affinities, ordegradation/turnover rate.

Parts of the BAG3-related nucleotide or aminoacid sequence may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites.

Reagents based on species homologs of BAG3 are considered equivalentrespect to the uses illustrated in the present invention.

The sequences falling within the scope of the present invention are notlimited to the specific sequences herein described, but also includeallelic variations thereof. The present invention will be illustrated bythe following examples, figures and tables which are not to beconsidered as limiting the scope of the invention.

DETAILED DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1—Expression of BAG3 mRNA and Protein in Primary cells fromleukemia patients. Leukemic cells were isolated from B-CLL patients'peripheral blood specimens by centrifugation through Ficoll-Hypaque (13)and cultured for 24 hours in RPMI 1640 medium supplemented with 10%fetal calf serum (10% FCS-RPMI), without or with fludarabine phosphate.A panel: cell mRNA was the extracted and BAG3 expression was verified byPCR (GAPDH expression is shown for comparative purpose); B panel: cellswere permabilised and analysed by indirect immunofluorescence with thepolyclonal antibody AC-BAG3-1. A=control rabbit Ig; b=cells incubatedwith control medium and analysed with anti-BAG3; c=cells incubated withfludarabine and analysed with anti-BAG3.

FIG. 2—Downmodulation of BAG3 protein levels by anti-BAG3 antisenseoligodeoxynucleotides. Leukemic cells were isolated from B-CLL patients'peripheral blood specimens by centrifugation through Ficoll-Hypaque andcultured for 20 hours without (b) or with BAG3 antisense (b+α) orcontrol nonsense (b+ν) phosphorothioate oligodeoxynucleotides (5 microM)described in the text. Then cells were permabilised and analysed byindirect immunofluorescence with the polyclonal antibody AC-BAG3-1.a=control rabbit Ig.

FIG. 3—Effect of anti-BAG3 antisense oligodeoxynucleotides onmitochondrial cytochrome c release in B-CLLs. Leukemic cells wereisolated from B-CLL patients' peripheral blood specimens bycentrifugation through Ficoll-Hypaque and cultured for the indicatedtimes without or with the BAG3 antisense or control nonsensephosphorothioate oligodeoxynucleotides (5 microM) described in the text.Then cell extracts were obtained and mitochondrial cytochrome c releasewas analysed according to ref. 8.

FIG. 4—Effect of anti-BAG3 antisense oligodeoxynucleotides on caspase 3activity in B-CLLs. Leukemic cells were isolated from B-CLL patients'peripheral blood specimens by centrifugation through Ficoll-Hypaque andcultured for the indicated times without or with the BAG3 antisense orcontrol nonsense phosphorothioate oligodeoxynucleotides (5 microM)described in the text. Then cell extracts were obtained and caspase 3activity was analysed according to ref. 9.

FIG. 5—Effect of anti-BAG3 antisense oligodeoxynucleotides on annexin Vbinding in B-CLLs Leukemic cells were isolated from B-CLL patients'peripheral blood specimens by centrifugation through Ficoll-Hypaque andcultured for 40 hours the indicated times without or with the BAG3antisense or control nonsense phosphorothioate oligodeoxynucleotides (5microM) described in the text. Then cell vitality was analysed bypropidium iodide incorporation in non permeabilized cells, while at thesame time annexin V binding was analysed by immunofluorescence accordingto ref. 10. A: percentages of alive, apoptotic and dead cells in thecytogram regions; B: PI—versus Annexin V—staining.

FIG. 6—Effect of anti-BAG3 antisense oligodeoxynucleotides on apoptosisin 15 B-CLL specimens Leukemic cells were isolated from B-CLL patients'peripheral blood specimens by centrifugation through Ficoll-Hypaque andcultured for 5 days without or with fludarabine phosphate (2 microgr/ml)and/or the BAG3 antisense or control nonsense phosphorothioateoligodeoxynucleotides (5 microM) described in the text. Then cellapoptosis was analysed by cell permeabilization and PI stainingaccording to ref. 11.

FIG. 7—Effect of anti-BAG3 antisense oliqodeoxvnucleotides on ALL cellapoptosis Leukemic cells were isolated from ALL patients' peripheralblood specimens by centrifugation through Ficoll-Hypaque and culturedfor four days (A panel) or the indicated times (B panel) without or withcytosine arabinoside (AraC, 1 microM) and/or the BAG3 antisense orcontrol nonsense phosphorothioate oligodeoxynucleotides (5 microM)described in the text. Then cell apoptosis was analysed by cellpermeabilization and PI staining according to ref. 11.

TABLE 1 Effect of anti-BAG3 antisense oligodeoxynucleotides on apoptosisin cells of the human osteosarcoma line SAOS. Table 1 Effect ofanti-BAG3 antisense oligodeoxynucleotides on apoptosis in cells of thehuman osteosarcoma line SAOS. Incubation Control Etoposide TopotecanOligo medium (5 microM) (40 ng/ml) 17.74* 38.32 36.84 BAG3 antisense52.38 73.40 68.62 control nonsense 25.84 45.40 41.60 *% of apoptosis

Cells of the SAOS line were incubated for 72 h in 10% FCS-RPMI withoutor with chemotherapeutic compounds (etoposide or topotecan) and/or theBAG3 antisense or control nonsense phosphorothioateoligodeoxynucleotides (5 microM) described in the text. Then cellapoptosis was analysed by cell permeabilization and PI stainingaccording to ref. 11.

FIG. 8—Effect of anti-BAG3 antisense oligodeoxynucleotides on BAG3Protein levels in cells of the human myeloid leukemia line U937 U937cells were cultured for 24 hours in 10% FCS-RPMI without or with theBAG3 antisense or control nonsense phosphorothioateoligodeoxynucleotides (5 microM) described in the text. Then cells werepermeabilised and analysed by indirect immunofluorescence withAC-BAG3-1.

FIG. 9—Effect of anti-BAG3 antisense oligodeoxynucleotides onstress-induced apoptosis in cells of the human myeloid leukemia lineU937 U937 cells were cultured for 40 h without or with diethylmaleate(DEM, 1.2 microM) and/or the BAG3 antisense or control nonsensephosphorothioate oligodeoxynucleotides (5 microM) described in the text.Then cell apoptosis was analysed by cell permeabilization and PIstaining according to ref. 11.

FIG. 10—Effect of anti-BAG3 antisense oligodeoxynucleotides onstress-induced apoptosis in normal human peripheral blood leucocytesLymphocytes (A panel) and monocytes (B panel) were obtained from humannormal peripheral blood specimens by centrifugation through aFicoll-Hypaque 50-72% density gradient and cultured in 10% FCS-RPMI for48 hours with or without a combination of DEM (1.2 microM) and 2ME (20microM) and/or the or the BAG3 antisense or control nonsensephosphorothioate oligodeoxynucleotides (5 microM) described in the text.Then cell apoptosis was analysed by cell permeabilization and PIstaining according to ref. 11.

TABLE 2 Protective effect of BAG3 hyperexpression on stress- inducedapoptosis in the human cell line 293. Transfected construct Incubation %of apoptosis Control pcDNA control medium  6.1 ± 0.3* DEM + 2ME 32.4 ±1.2 BAG3-pcDNA control medium  5.3 ± 0.2 DEM + 2ME 13.4 ± 0.5 *mean ofduplicates ± SD

Cells of the human line 293 were transfected using a Fugene (Roche)preparation with a pcDNA construct hyperexpressing BAG3 or a voidcontrol pcDNA. BAG3 protein hyperexpression was verified byimmunofluorescence. Then the cells were incubated for 48 hours in 10%FCS-RPMI with or without a combination of DEM+2ME and apoptosis wasanalysed by cell permeabilization and PI staining according to ref. 11.

TABLE 3 BAG3 expression influences the growth of human neoplastic(osteosarcoma) cells xenografted in nude mice # mouse tumour volume(mm³) <40 B 65 C <40 2 A <40 B 45 C <40 3 A <40 B .92 C <40 4 A <40 B<40 C <40 5 A <40 B 65 C <40 Human osteosarcoma cells of the SaOs line(10 × 10⁶), wild type (A) or stably transfected with aBAG3-hyperexpressing (B) or a control void (C) vector, were injected in6 week-old nu/nu mice; tumour volume was measured every week. Finalresults at the end of the 8th week are reported.

FIG. 11 Expression of BAG3 Protein in ALL cells and its downmodulationby BAG3-specific antisense oligonucleotides. A—ALL cells (1×10⁶/ml) werecultured in 10% FCS-RPMI without or with control nonsense(TTATATTCTATTATATTTATGMCTCC, SEQ ID NO 12, nonsense 1) or BAG3-specificantisense (TGCATCATGGGCGAGTGGGTGGCGG, SEQ ID NO 9, antisense 1)oligonucleotides (5 microM) for 24 hr. Then cell lysates were obtainedand analyzed in Western blot with anti-BAG3 (AC-BAG3-1; analogousresults were obtained with AC-BAG3-2) or anti-tubulin antibodies. B—ALLcells (1×10⁶/ml) were cultured in 10% FCS-RPMI without or withBAG3-specific antisense (TGCATCATGGGCGAGTGGGTGGCGG, SEQ ID NO 9,antisense 1) (a) or control nonsense (TTATATTCTATTATATTTATGMCTCC, SEQ IDNO 12, nonsense 1) (b) oligonucleotides (5 microM) for 24 hr. Then thecells were analyzed by intracellular immunofluorescence with theanti-BAG3 polyclonal antibody. Negative controls with a control rabbitantibody preparation are shown on the left in a and b. Results arerepresentative of experiments with at least three different ALL samples;comparable results were obtained using any one of the three antisense ornonsense ODN.

FIG. 12—Effects of BAG3-specific antisense oligonucleotides or AraC onALL cell apoptosis. A—ALL cells (1×10⁶/ml) from ten different sampleswere cultured in 10% FCS-RPMI without or with control nonsense orBAG3-specific antisense oligonucleotides (5 microM), or with AraC (10microM), for 4 days. Then cell apoptosis was analyzed by propidiumiodide incorporation in permeabilized cells and flow cytometry.Student's t test was performed to evaluate the difference betweenapoptosis percentages detected in control and BAG3 antisense-culturedcells, respectively.

TABLE 4 Binding of hybridoma mother clone supernatants to MAP-BAG3constructs as detecteds by ELISA test. Map 1 Map 2 Map 3 Map 4 Map 1 Map2 Map 3 Map 4 Map 1 Map 2 Map 3 Map 4 1/2  1.254 2.475 0.050 0.042 1.8080.504 0.412 0.424 3.825 0.054 0.053 0.050 1/10 0.345 0.966 0.047 0.0420.474 0.137 0.128 0.123 3.756 0.053 0.050 0.046 1/2  2.012 1.568 0.0470.042 1.782 0.666 0.438 0.451 3.747 0.059 0.065 0.062 1/10 0.715 0.4600.045 0.042 0.608 0.164 0.150 0.149 3.729 0.046 0.051 0.049 1/2  0.0440.048 0.300 0.046 2.133 0.646 0.547 0.396 3.822 0.047 0.047 0.052 1/100.042 0.046 0.109 0.045 0.580 0.154 0.140 0.138 3.556 0.049 0.046 0.0481/2  AC-4 AC-1 AC-7 1/10 1/2  AC-5 AC-2 AC-8 1/10 1/2  AC-6 AC-3 AC-91/10 ELISA test of antibodies produced by the monoclonal mother clonesAC-1, AC-2, AC-3, AC-4, AC-5, AC-6, AC-7, AC-8, AC-9. Supernatants wereobtained from nine hybridoma mother clones (AC-1 to -9) and analysed fortheir binding to MAP-BAG3 constructs.

FIG. 13 Binding of BAG3-specific polvclonal and monoclonal antibodies toproteins from HeLa or Primary acute leukemia cells. A—Lysates from HeLaor primary acute leukemia cells were analysed in Western blotting usingAC-BAG3-1 (central lanes: 3 and 4) or AC-BAG3-2 (lanes 1, 2 and 5)antibodies (A). B—Supernatants from the hybridoma mother clones AC-1(1), AC-2 (2), AC-3 (3) or AC-4 (4) were analysed for their binding toproteins from HeLa cells in Western blotting.

REFERENCES

-   1. Nicholson D W. 2000. From bench to clinic with apoptosis-based    therapeutic agents. Nature 407: 810.-   2. Takayama S and Reed J C. 2001. Molecular chaperone targeting and    regulation by BAG family proteins. Nature Cell Biology 3: E237.-   3. Takayama T, Xie Z, and Reed J C. 1999. An evolutionarily    conserved family of Hsp70/Hsc70 molecular chaperone regulators. J    Biol Chem 274: 781.-   4. Doong H, Price J, Kim Y-S, Gasbarre C, Probst J, Liofta L A,    Blanchette J, Rizzo K, and Khon E. 2000. CAIR-1/BAG-3 forms and    EGF-regulated ternary complex with phospholipase C gamma and    Hsp70/Hsc70. Oncogene 19: 4385.-   5. Lee J-H, Takahashi T, Yasuhara N, Inazawa J, Kamada S,    Tsujimoto Y. 1999. Bis, a Bcl-2-binding protein that synergize with    Bcl-2 in preventing cell death. Oncogene 18: 6183.-   6. Liao 0, Ozawa F, Friess H, Zimmermann A, Takayama S, Reed J C,    Kleeff J, Buchler M W. 2001. The anti-apoptotic protein BAG-3 is    overexpressed in pancreatic cancer and induced by heat stress in    pancreatic cancer cell lines. FEBS Left. 503:151.-   7. Antoku K, Maser R S, Scully W J Jr, Delach S M, and Johnson    D E. 2001. Isolation of Bcl-2 binding proteins that exhibit homology    with BAG-1 and Suppressor of Death Domains protein. Biochem Biophys    Res Comm 286: 1003.-   8. Renz A, Berdel W E, Kreuter M, Belka C, Schulze-Osthoff K,    Los M. 2001. Rapid extracellular release of cytochrome c is specific    for apoptosis and marks cell death in vivo. Blood 98:1542.-   9. Kluck R M, Martin S J, Hoffman B M, Zhou J S, Green D R and    Newmeyer D D. 1997. Cytochrome c activation of CPP32-like    proteolysis plays a critical role in a Xenopus cell-free apoptosis    system. EMBO J. 16: 4639.-   10. Koopman G, Reutelingsperger C P, Kuijten G A, Keehnen R M, Pals    S T, van Oers M H. 1994. Annexin V for flow cytometric detection of    phosphatidylserine expression on B cells undergoing apoptosis.    Blood. 84:1415.-   11. Nicoletti I, Migliorati G, Pagliacci M C, Grignani F, Riccardi    C A. 1991. A rapid and simple method for measuring thymocyte    apoptosis by propidium iodide staining and flow cytometry. J Immunol    Methods 139: 271.-   12. Tassone P, Tuccillo F, Bonelli P, Turco M C, Cecco L, Cerra M,    Bond H M, Barbieri V, Venuta S. 1998. CD36 is rapidly and    transiently upregulated on phytohemagglutinin (PHA)-stimulated    peripheralblood lymphocytes. Analysis by a new monoclonal antibody    (UN7). Tissue Antigens 51: 671.-   13. Romano M F, Lamberti A, Tassone P, Alfinito F, Costantini S,    Chiurazzi F, Defrance T, Bonelli P, Tuccillo F, Turco M C,    Venuta S. 1998. Triggering of CD40 antigen inhibits    fludarabine-induced apoptosis in B-CLL cells. Blood 92: 990.-   14. Manolagas S C. 2001. Manipulating programmed cell death for    better living. Sci STRKE 19: 87.-   15. Drissi R, Zindy F, Roussel M F, Cleveland J L. c-Myc-mediated    regulation of telomerase activity is disabled in immortalized    cells. 2001. J Biol Chem 276: 29994.-   16. Petit-Frere C, Capulas E, Lyon D A, Norbury C J, Lowe J E,    Clingen P H, Riballo E, Green M H, Arlett C F. 2000. Apoptosis and    cytokine release induced by ionizing or ultraviolet B radiation in    primary and immortalized human keratinocytes. Carcinogenesis 21:    1087.-   17. Brezden C B, Rauth A M. 1996. Differential cell death in    immortalized and non-immortalized cells at confluency. Oncogene 12:    201.-   18. Iordanov M S, Wong J, Newton D L, Rybak S M, Bright R K, Flavell    R A, Davis R J, Magun B E. 2000. Differential requirement for the    stress-activated protein kinase/c-Jun NH(2)-terminal kinase in    RNAdamage-induced apoptosis in primary and in immortalized    fibroblasts. Mol Cell Biol Res Commun 4:122.-   19. Marsden V S, Strasser A. 2000. Control of Apoptosis in the    Immune System: Bcl-2, BH3-Only Proteins and More. Annu Rev Immunol    2002, October 16.-   20. Roth W, Grimmel C, Rieger L, Strik H, Takayama S, Krajewski S,    Meyermann R, Dichgans J, Reed J C, Weller M. 2000. Bag-1 and Bcl-2    gene transfer in malignant glioma: modulation of cell cycle    regulation and apoptosis. Brain Pathol. 10: 223.-   21. Zong W X, Lindsten T, Ross A J, MacGregor G R, Thompson    C B. 2001. BH3-only proteins that bind pro-survival Bcl-2 family    members fail to induce apoptosis in the absence of Bax and Bak.    Genes Dev 15:1481.-   22. Gewirtz A M. 1999. Oligonucleotide therapeutics: clothing the    emperor. Curr Opin Mol Ther 3: 297.-   23. Opalinska J B, Gewirtz A M. 2002. Nucleic-acid therapeutics:    basic principles and recent applications. Nat Rev Drug Discov 7:    503.-   24. Keah H H, Kecorius E, Hearn M T. 1988. Direct synthesis and    characterisation of multi-dendritic peptides for use as immunogens.    J Pept Res 51: 2.-   25. Tam J P. 1988. Synthetic peptide vaccine design: synthesis and    properties of a high-density multiple antigenic peptide system. Proc    NatI Acad Sci USA 85: 5409.-   26. Ota S, Ono T, Morita A, Uenaka A, Harada M, Nakayama E. 2002.    Cellular processing of a multibranched lysine core with tumor    antigen peptides and presentation of peptide epitopes recognized by    cytotoxic T lymphocytes on antigen-presenting cells. Cancer Res    62:1471.

1.-29. (canceled)
 30. A method for modulating apoptosis in a cell byregulating BAG-3 expression, which comprises using isolatedpolynucleotides or oligonucleotides that recognize or modulate BAG3protein expression and wherein said polynucleotides or oligonucleotidesare selected from the group of nucleic acid sequences consisting of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, and fragments and a complementary sequence thereof.31. The method according to claim 30, wherein said isolatedpolynucleotides or oligonucleotides have at least 90% homology to BAG3SEQ ID NO:1 or fragments or a complementary sequence thereof.
 32. Themethod according to claim 30, wherein said cell is a primary cell. 33.The method according to claim 30, wherein said polynucleotides oroligonucleotides consist of SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11 ora sequence encoding a peptide of SEQ ID NO:15, SEQ ID NO:6, SEQ ID NO:17or SEQ ID NO:18.
 34. The method according to claim 30, wherein saidisolated polynucleotides or oligonucleotides that recognize or modulateBAG3 protein expression are comprised in a vector.
 35. The methodaccording to claim 30 comprising genetically engineering a host cellwith isolated polynucleotides or oligonucleotide/s that recognize ormodulate BAG3 protein expression, said polynucleotides oroligonucleotides being selected from the group of nucleic acid sequencesconsisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:10, and SEQ ID NO:11.
 36. The method according toclaim 35, wherein said polynucleotides or oligonucleotides are inoperative association with a regulatory sequence that controlsexpression of said polynucleotides or oligonucleotides in the host cell.37. The method according to claim 31, wherein said isolatedpolynucleotides or oligonucleotides with at least 90% homology to BAG3SEQ ID NO:1 or fragments or a complementary sequence thereof, encode apeptide with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, or SEQ ID NO:18 and modulate apoptosis in primary cells.
 38. Atherapeutic method comprising the method for modulating apoptosis in acell according to claim 30 for the treatment of a disease involvingexcessive or defective apoptosis, said therapeutic method comprisingadministering to a subject in need thereof an effective amount of apolynucleotide or oligonucleotide selected from the group of nucleicacid sequences consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, or a sequenceencoding a peptide of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ IDNO:18 and fragments and complementary sequences thereof.
 39. Thetherapeutic method according to claim 38, wherein said disease involvingexcessive or defective apoptosis is selected from the group consistingof primary leukemias, acute or chronic tissue damage, such as heart,kidney, brain or other organ ischaemia, HIV-related damage of brain orother tissues, skeletal muscle disorders, transplantation rejection;chronic degenerative disorders such as Parkinson's disease, amyotrophiclateral sclerosis and others; and neoplastic, autoimmune and otherdiseases involving excessive or defective apoptosis; tissue repair orwound healing, treatment of surgical incisions, and ulcers, such asstomach or diabetic ulcers.
 40. The therapeutic method according toclaim 38, wherein said subject in need thereof is under chemotherapeutictreatment.
 41. A method for detecting the presence of the nucleotidesequence SEQ ID NO:1 or parts thereof in a sample, said methodcomprising the steps of contacting the sample with a polynucleotide oroligonucleotide that binds to it and forms a complex with the nucleotideor parts thereof under conditions sufficient to form the complex, anddetecting said complex.
 42. The method according to claim 41, whereinsaid part of SEQ ID NO:1 to which said polynucleotide or oligonucleotidebinds is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, oris a sequence encoding a peptide of SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17 and SEQ ID NO:18.
 43. A kit for identifying or diagnosing adisease involving excessive or defective apoptosis comprising at leastone polynucleotide or nucleotide selected from the group consisting ofSEQ ID NOS:1, 3, 5, 7, 9, 10 and 11 or a sequence encoding a peptide ofSEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 fragments anda complementary sequence thereof.